Fish vaccine

ABSTRACT

A salmonid alphavirus polypeptide comprising an epitope capable of inducing a virus neutralizing immune response, nucleic acids encoding the polypeptide, a vaccine comprising the polypeptide and a method of producing salmonid alphavirus neutralizing antibodies.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Divisional Application of U.S. Ser. No. 12/066,868filed Mar. 14, 2008, now U.S. Pat. No. 7,794,724 issued Sep. 14, 2010,which is the National Phase entry under 35 U.S.C. 371 of InternationalApplication No. PCT/EP2006/066401 filed Sep. 15, 2006, which claimspriority to European Patent Application No.: 05020223.3 EP filed Sep.16, 2005.

The present invention relates to veterinary immunology, namely to theimmunological response of fish to a virus. More specifically, theinvention provides an epitope of salmonid alphaviruses which epitope iscapable of inducing a virus neutralising immune response.

In particular the invention relates to a polypeptide comprising acertain amino acid sequence, a protein comprising such polypeptide, to acarrier comprising such protein, and to a method of producingantibodies. Further the invention relates to a nucleic acid encodingsuch polypeptide or such protein, and to a carrier comprising such anucleic acid. Also, the invention relates to a vaccine and a diagnostickit comprising such a polypeptide, protein, carrier, or nucleic acid.

The fish viruses salmon pancreas disease virus (SPDV) and sleepingdisease virus (SDV) have been described to belong to the genusalphavirus, in the family Togaviridae et al., 2000, J. of Virol., vol.74, p. 173-183). The two aquatic viruses are closely related, and form agroup that was found to be separate from the groups around theSindbis-like and encephalitis-type alphaviruses (Powers et al., 2001, J.of Virol., vol. 75, p. 10118-10131). Therefore, they have beenclassified as variants of the species salmonid alphavirus (SAV) (Westonet al., 2002, J. of Virol., vol. 76, p. 6155-6163).

SDV has been isolated from trout in France and the United Kingdom (UK),and SPDV from salmon in Ireland and the UK. Also in trout and salmonfrom Norway, SPDV-like viruses have been isolated. Through genomiccharacterisation, these Norwegian isolates however were found to be adistinct subgroup. Therefore a subdivision of the SAV species has beenproposed very recently (Weston et al., 2005, Dis. of Aq. Organ., vol.66, p. 105-111; Hodneland et al., 2005, Dis. of Aq. Organ., vol. 66, p.113-120), wherein SAV subtype 1 is formed by the viruses havingresemblance to the SPDV isolates from Ireland and UK; subtype 2 isformed by the SDV isolates; and subtype 3 is formed by the Norwegianisolates of SPDV.

The three SAV subtypes are characterised by their reference strains:

-   -   for subtype 1: SPDV isolate F93-125; the genomic sequence is        available under GenBank accession number: AJ316244, and its        envelope protein 2 (E2) as: CAC87722.    -   for subtype 2: SDV isolate S49P; the genomic sequence is        available under GenBank accession number: AJ316246, and its E2        protein as: CAB59730, amino acids 357-794.    -   for subtype 3: SPDV isolate N3; the genomic sequence is        available under GenBank accession number: AY604237, and its E2        protein as: AAU01400, amino acids 353-790.        The three SAV subtypes all cause a serious disease in salmon and        trout, though the specific symptoms and their severity may vary        with the subtype and the fish species. The various isolates are        cross-protective to some extent, and antibodies to one of the        subtypes show cross-reaction with the other subtypes.

A comprehensive overview of fish vaccines for aquaculture is fromSommerset et al., (2005, Expert Rev. Vaccines, vol. 4, p. 89-101). Theaquaculture industry producing salmon or trout, suffers considerablyfrom outbreaks of SAV, which cause reduced growth, and mortality ofbetween 10-60 percent of the animals. Research has led to thedevelopment of vaccines (EP 712,926), and NORVAX COMPACT PD, aninactivated SAV subtype 1 virus vaccine, is now available commerciallyfor immunisation of salmon against SPDV. Vaccines based on SAV viralproteins have also been described (EP 1,075,523).

These described SAV vaccines although effective, however require carefulselection of a process for the inactivation of the virus that does notdiminish the viral immunogenicity. Also meticulous quality control ofthe viral inactivation process is required, to assure no live virusremains. The same is true for subunit vaccines based on whole proteinsfrom a virus that are isolated from viral cultures. Consequently,production of such vaccines comes at a certain price.

An improvement is the production of subunit vaccines in a recombinantexpression system, where no pathogenic virus is used. Nevertheless, theexpression of the often large viral proteins is a heavy burden on thecapacity of the expression system, making expression less effective.Also this is inefficient, as most of the protein expressed does notattribute to the actual immune-activation. On the contrary, manyantigens possess immunodominant regions that are not involved inimmuneprotection, but may even mask regions that are immuneprotective.As a result, immunisation with protein comprising such regions iscounter-effective: an organism's humoral and/or cellular immunesystem isactivated against these parts of the antigen but this does not interferewith the infection or the replication by the pathogen, or with thesymptoms of disease.

Consequently there is a need for alternative SAV vaccines that areimproved and more efficient, in that they are based on small butrelevant immunogenic parts of the SAV viral proteins per se: theprotective epitopes.

As is well known, the stimulation of an organism's immune system throughT- and B-lymphocytes is based on the molecular recognition of an epitopeby the T- or B-cell receptor. If epitopes are linear, they exist on anantigen as a continuous, sequential structure, and are relatively small;e.g. for protein, regions of 8-15 amino acids can be bound by MHC I orII molecules for presentation to the lymphocyte receptors (reviewed e.g.by Germain & Margulies, 1993, Annu. Rev. Immunol., vol. 11, p. 403-450).However, epitopes may also be formed as a result of the 3-dimensionalfolding of an antigen, such epitopes are discontinuous, and areassembled from e.g. amino acids that are not sequential in the protein'samino acid sequence. As a result, the region of an antigen spanning adiscontinuous epitope is commonly larger than that of a linear epitope.

An epitope is protective if it can induce an immune response that iscapable to effectively interfere with the extent or the progression ofinfection or disease.

Of all possible protective epitopes, either linear or conformational,those that cause virus neutralisation (VN) are most effective; suchepitopes induce a humoral immune response that neutralises viralinfectivity, and/or a cellular immune response that causes lysis ofvirus infected cells. Virus neutralisation is for instance effectuatedby preventing viral attachment and/or entry into host cells.

Viral VN epitopes are commonly located on molecules involved inessential viral processes, such as for attachment or entry of hostcells. Therefore their presence is relevant to the virus, leading totheir conservation. This makes VN epitopes highly effective in vaccines,as well as in diagnostic assays.

The identification of an epitope in an antigen is a complex process,even though modern techniques can sometimes assist in their prediction:computer driven prediction can provide an indication of relevant areas.Such predictions use algorithms describing shape and charge of aprotein, such as developed by Hopp & Woods (1981, Proc. Natl. Acad. Sci.USA, vol. 78, p. 3824-3828), and Chou & Fassman (1978, Advances inEnzymology, vol. 47, p. 45-148). However, such predictions are almostexclusively of use to linear epitopes. Computer prediction ofdiscontinuous epitopes with some level of accuracy has been described(Kulkarni-Kale et al., 2005, Nucl. Acids Res., vol. 33, p. W168-W171),but this technique is only applicable to proteins of which the 3Dcrystal structure has been determined. Critical to all predictionmethods is to follow up by verification whether the predicted epitope isat all immunogenic, and has protective capacity.

Similarly, the so-called Pepscan technique can be used to identifylinear epitopes by an automated screening assay (Geysen et al., 1984,Proc. Natl. Acad. Sci. USA, vol. 81, p. 3998-4002). Again, this is of nouse to discontinuous epitopes.

Mapping and characterisation of linear epitopes of alphaviruses has beendescribed, e.g. for Semliki forest virus E2: the regions from amino acid(aa) 227-243, and 297-310 (Grosfeld et al., 1991, Vaccine, vol. 9, p.451-456); regions 166-185, and 286-305 (Ariel, et al., 1990, Arch.Virol. vol. 113, p. 99-106); and region 297-352 (Grosfeld, et al., 1992,J. of Virol., vol. 66, p. 1084-1090).

Epitopes in alphaviral proteins that can induce a VN immune responsehave also been described: aa 170-220 of Sindbis virus E2 protein(Strauss & Strauss, 1994, Microbiol. Reviews, vol. 58, p. 491-562).Pence et al. (1990, Virology, vol. 175, p. 41-49), describe adiscontinuous VN epitope of Sindbis virus E2 protein: E2c, covering thearea of amino acids 62-159.

However, due to the low sequence homology between SAV and the otheralphaviruses, none of these epitopes could be located on the SAVproteins. Homologies between structural protein sequences of SAV andother alphaviruses are in the order of 31-33% (Weston, 2002, supra).

For SAV itself, VN antibodies and their use in a serological assay havebeen described (Graham et al., 2003, J. of Fish Dis., vol. 26, p.407-413), but these were polyclonal antibodies. Although effective inthe assay described, such antibodies are not useful in theidentification of a specific VN epitope, as such antibodies in an animalserum cover a wide variety of epitopes.

For this purpose of identifying VN epitopes on viral proteins,monoclonal antibodies are required. Although monoclonal antibodiesagainst SAV proteins have been described (e.g. Graham et al., 2003,supra), these were not virus neutralising.

Consequently, so far no VN epitope of SAV viral proteins has beendescribed. Nevertheless, for use in the fields of vaccination anddiagnostics for SAV, it would be highly advantageous to have a VNepitope of an SAV viral protein.

It is therefore an object of the invention to provide for the first timea virus neutralising epitope from a salmonid alphavirus protein thatallows for development of vaccines and diagnostics that improve on theefficacy and specificity of those known so far.

Surprisingly it was found now, that a polypeptide covering the aminoacid region between aa 158 and 252 of the SAV E2 protein, incorporates aconformational, virus neutralising epitope of SAV E2 protein.

This VN epitope can now be employed for the generation of effectivevaccines and diagnostic assays. The major advantage being that the VNepitope is very small in relation to the complete SAV E2 protein, whichis 438 aa in size, while still incorporating the essential immunogenicportion of the SAV E2 protein.

This has a number of advantageous effects: the expression of the SAV E2VN epitope in a recombinant expression system is much more efficientthan that of the complete SAV E2, by not needlessly weighing on thecapacity and the resources for expression of a bulk of protein that isnot directly related to providing the essential immune response. Inquantitative terms the efficiency of expression of the VN epitope overthe full E2 is improved by 4.6 times (95 amino acids versus 438);additionally a qualitative improvement is reached: by not overloadingthe expression system's capacity for expression and post-translationalprocessing, and therefore not inducing premature cell-lysis andsubsequent proteolysis, the VN epitope protein produced is of betterquality than the E2 protein would be.

Put in another way, when samples of SAV E2 VN epitope protein and offull length SAV E2 protein of the same amount are compared, then theimmunogenic potency of the sample of VN epitope protein is more than 5times higher than that of E2 protein. This is a surprising and highlyrelevant improvement of the efficiency of expression of SAV E2 subunits.

Another important advantage resulting from the small size of the SAV E2VN epitope of the invention, is its improved possibility for beingexpressed by a live recombinant carrier (LRC); such LRC's, replicate inthe vaccinated host, and often can only incorporate a limited amount offoreign nucleic acid into their genome. Advantageously, a nucleic acidencoding the VN epitope of the invention which measures less than 300nucleotides, can be incorporated in most such live recombinant carriers,whereas a nucleic acid encoding the full length E2 protein, measuringover 1300 nucleotides, gives rise to problems in replication,transcription, and assembly and e.g. diminishes the replicative capacityof the LRC.

Alternatively, when an LRC does allow for larger foreign inserts, thesmall size of the SAV E2 VN epitope now allows for insertion of a numberof inserts. Such multiple inserts can be inserted in fusion, that is allbehind one promoter, or each insert with its own promoter.

Improved efficiency of such an LRC is very relevant for vaccination inaquaculture: because of the animal husbandry methods employed, and thesheer number of animals to be vaccinated, individual handling of animalsis impratical and very laborious. Therefore, the use of aself-replicating immunogen, such as an LRC which makes efficientmass-vaccination procedures possible, is an important efficiencyimprovement.

Therefore, in a first aspect, the invention relates to a polypeptidecomprising an amino acid sequence having at least 90% amino acididentity to any one of SEQ ID NO: 1-3 in a region corresponding to saidSEQ ID NO, characterised in that the polypeptide is at least 95 and atmost 166 amino acids in size.

SEQ ID NO: 1-3 represent the aa region 158-252 of SAV E2 protein of thereference strains: F93-125, S49P, and N3 respectively.

For the invention expressions indicating an amino acid sequence regionof the SAV E2 protein, such as 158-252 are to be interpreted to mean:starting with aa 158, up to and including aa 252.

By incorporating one of the sequences of SEQ ID NO: 1-3, the polypeptideof the invention comprises the region of amino acids 158-252 of SAV E2protein, which region incorporates the discontinuous VN epitope of SAVE2 protein.

The length and position of the SEQ ID NO's described herein is presentedgraphically in FIG. 1, relative to the full length SAV E2.

For the invention, such amino acid alignment must be determined with thecomputer program “BLAST 2 SEQUENCES” by selecting sub-program: “BlastP”(Tatusova & Madden, 1999, FEMS Microbiol. Letters, vol. 174, p. 247-250.The comparison-matrix to be used is: “Blosum62”, with the defaultparameters: open gap penalty: 11; extension gap penalty: 1, and gapx_dropoff: 50. This computer program reports the percentage of aminoacids that are identical, that is counting only exact matches, as“Identities”.

For the invention, such amino acid alignment must be determined with thecomputer program “BLAST 2 SEQUENCES” by selecting sub-program: “BlastP”(Tatusova & Madden, 1999, FEMS Microbiol. Letters, vol. 174, p. 247-250.The comparison-matrix to be used is: “Blosum62”, with the defaultparameters: open gap penalty: 11; extension gap penalty: 1, and gapx_dropoff: 50. This computer program reports the percentage of aminoacids that are identical, that is counting only exact matches, as“Identities”.

SAV E2 fragments containing this region of aa 158-252 of SAV E2, werefound to contain the VN epitope of the invention. This is evident fromthe experimental results obtained; the positive VN scores are alsoindicated in FIG. 1, and these and other results are outlined in theexperimental section.

It was found now that upon multiple alignment of the amino acid region158-252 from many SAV E2 proteins, comprising isolates from each of thethree SAV subtypes, the level of amino acid identity found variedbetween 90 and 100%. This is represented in Table 1 below, and in FIG.2. This very high level of conservation indicates that variations inamino acid sequence identity of 90%, are within the natural variation ofthe amino acid sequences that are found in this region of the E2 proteinof SAV. Therefore proteins having amino acid identities within thisrange of 90-100% to polypeptides according to the invention, apparentlyare natural variants and therefore are within the scope of theinvention.

TABLE 1 Percentage amino acid identity of amino acid regions 158-252from SAV E2 proteins compared to one reference sequence: % aa identityAccess. nr. to reference SAV type SAV isolate SEQ ID NO: AAU01400.1reference 3 N3 3 AAU01398.1 100 3 CMS1 AAU01396.1 100 3 Hav1 AAU01402.197 3 Tun1 NP_740641.1 92 1 NP_647497.1 92 1 CAC87722.1 92 1 F93-125 1CAB42823.1 92 1 CAC87661.1 90 2 NP_598185.1 90 2 S49P 2 CAB59730.1 90 2NP_740659.1 90 2

Results were determined using the ALIGN+ PROGRAM (SE Central), withsettings to “global alignment against a reference sequence”, using exactmatches, a scoring matrix=Blosum 62, and other parameters set at defaultvalue. The reference sequence was that of SAV isolate N3 (acc. nr:AAU01400.1)

In a preferred embodiment, the polypeptide according to this aspect ofthe invention comprises an amino acid sequence having at least 91% aminoacid sequence identity to any one of SEQ ID NO: 1-3. More preferred is apolypeptide having 92, 93, 94, 95, 96, 97, 98, 99, or even 100% aminoacid sequence identity to any one of SEQ ID NO: 1-3, in that order ofpreference.

Preferably the size for a polypeptide according to this aspect of theinvention does not exceed 166 amino acids. This corresponds to thelength of the region from aa 139-304 of SAV E2 protein.

As a result, polypeptides according to this aspect of the invention maybe derived from the SAV E2 protein from anywhere between aa 87 (beingposition 252 minus 166) and aa 323 (being position 158 plus 166),provided they have a maximum length of 166 amino acids. Suchpolypeptides still incorporate an amino acid sequence regioncorresponding to the SAV E2 region of aa 158-252 which contains the VNepitope according to the invention, and because of the natural variationthat exists in SAV E2 sequences in this region, their percentage ofamino acid identity to SEQ ID NO: 1-3 is between 90 and 100%.

Techniques to obtain the polypeptides according to the invention arewell known in the art. Preferably genetic engineering techniques andrecombinant DNA expression systems are employed to express exactly thedesired fragments.

The nucleic acid sequences that can be used to encode a polypeptideaccording to the invention are described herein and/or are publiclyavailable. Such sequences can be obtained, manipulated and expressed bystandard molecular biology techniques that are well-known to the skilledartisan, and that are explained in great detail in standard text-bookslike: Molecular cloning: a laboratory manual (Sambrook & Russell: 2000,Cold Spring Harbor Laboratory Press; ISBN: 0879695773), and: Currentprotocols in molecular biology (Ausubel et al., 1988+ updates, GreenePublishing Assoc., New York; ISBN: 0471625949).

To construct a nucleic acid encoding a polypeptide according to theinvention, preferably DNA fragments in the form of plasmids areemployed. Such plasmids are useful e.g. for enhancing the amount of aDNA-insert, for use as a probe, and as tool for further manipulations.Examples of such plasmids for cloning are plasmids of the pET, pBR, pUC,pGEM, and pcDNA plasmid series, all these are readily available fromseveral commercial suppliers.

To obtain the desired polypeptide, the proper nucleic acid sequences areconstructed e.g. by using restriction enzyme digestion, by site directedmutations, or preferably by polymerase chain reaction (PCR) techniques.Standard techniques and protocols for performing PCR are for instanceextensively described in: PCR primers: a laboratory manual (Dieffenbach& Dveksler, 1995, CSHL Press, ISBN 879694473). For instance, byselecting a PCR primer that hybridises at a particular place on an SAVE2 encoding gene, rec DNA fragments are produced that encodepolypeptides starting or ending at the desired amino acid of the SAV E2.

For the purpose of cloning, protein purification, detection, orimprovement of expression level, additional sequences may be added,preferably already incorporated in the PCR-primers used.

The DNA encoding the polypeptide according to the invention can e.g. becloned from the PCR product into an expression vector. In suchexpression vectors, the nucleic acid encoding the desired fragment ofSAV E2 is operably linked to a transcriptional regulatory sequence suchthat it is capable of controlling the transcription of the nucleic acidsequence. Suitable expression vectors are, amongst others, plasmids,cosmids, viruses and YAC's (Yeast Artificial Chromosomes) which comprisethe necessary control regions for replication and expression.

Transcriptional regulatory sequences are well-known in the art andcomprise i.a. promoters and enhancers. Those skilled in the art are wellaware that the choice of a promoter extends to any eukaryotic,prokaryotic or viral promoter capable of directing gene transcription,provided that the promoter is functional in the expression system thatis to be used.

Transcriptional regulatory sequences that are suitable for use in anexpression DNA plasmid comprise promoters such as the (human)cytomegalovirus immediate early promoter and the Rous sarcoma virus LTRpromoter (Ulmer et al., 1993, Science, vol. 259, p. 1745-1748), and themajor late promoter of Adenovirus 2 and the β-actin promoter (Tang etal., 1992, Nature, vol. 356, p. 152-154). The regulatory sequences mayalso include terminator and polyadenylation sequences, such as the wellknown bovine growth hormone polyadenylation sequence, the SV40polyadenylation sequence, and the human cytomegalovirus terminator andpolyadenylation sequences.

Bacterial, yeast, fungal, insect, and vertebrate cell expression systemsare used very frequently. Such expression systems are well-known in theart and generally available, e.g. through Invitrogen (the Netherlands).

A host cell used for expression of a polypeptide or protein (as outlinedbelow) according to the invention, may be a cell of bacterial origin,e.g. from Escherichia coli, Bacillus subtilis, Lactobacillus sp., orCaulobacter crescentus, or the aquatic bacteria Yersinia ruckeri, andVibrio anguillarum, all in combination with the use of bacteria-derivedplasmids or bacteriophages for expressing the sequence encoding thepolypeptide or protein (as outlined below) according to the invention.The host cell may also be of eukaryotic origin, e.g. yeast-cells (e.g.Saccharomyces or Pichia) in combination with yeast-specific vectormolecules; insect cells in combination with recombinant baculo-viralvectors e.g. Sf9 and pVL1393 (Luckow et al., 1988, Bio-technology, vol.6, p. 47-55); plant cells, in combination with e.g. Ti-plasmid basedvectors or plant viral vectors (Barton, et al., 1983, Cell, vol. 32, p.1033-1043); or mammalian cells also with appropriate vectors orrecombinant viruses, such as Hela cells, CHO, CRFK, or BHK cells, orfish cells such as Chinook salmon embryo (CHSE-214) cells, Atlanticsalmon cell lines and Rainbow trout cell lines.

Next to these expression systems, expression in organisms such as algae,or plants, are attractive expression systems, as these can beincorporated into the feed, and be used as oral vaccine. By feeding suchmaterials to fish to be vaccinated, efficient mass vaccination isachieved.

The technique of in vivo homologous recombination, well-known in theart, can be used to introduce a recombinant nucleic acid sequence intothe genome of a bacterium, virus, or organism of choice.

Expression may also be performed in so-called cell-free expressionsystems. Such systems comprise all essential factors for expression ofan appropriate recombinant nucleic acid, operably linked to a promoterthat is capable of expression in that particular system. Examples arethe E. coli lysate system (Roche, Basel, Switzerland), or the rabbitreticulocyte lysate system (Promega corp., Madison, USA).

In an other embodiment, the invention relates to a protein comprisingthe polypeptide of the invention, whereby said protein does not comprisea part of an SAV E2 protein comprising said polypeptide and being morethan 166 amino acids in size.

Such proteins according to the invention are fusion- and carrierproteins comprising the polypeptide according to the invention.

A fusion or carrier protein is formed through an assembly of two or morestrands of amino acids, giving a combination that does not occurnaturally. The strands can be of equal or different length. Thecombination of the strands can be accomplished by several means, e.g.:

-   -   chemically; by coupling, conjugation or cross-linking, through        dehydration, esterification, etc., of the amino acid sequences        either directly or through an intermediate structure.    -   physically; by coupling through capture in or on a        macromolecular structure    -   by molecular-biological fusion; through the combination of        recombinant nucleic acid molecules which comprise fragments of        nucleic acid capable of encoding each of the two, such that a        single continuous expression product is finally produced.

Techniques for applying these couplings and fusions are well known inthe art, and are for instance described in the handbooks of Sambrook &Russel, and of Ausubel, as described supra.

The use of such fusion- or carrier-proteins has several advantages, forinstance:

-   -   easier handling; for example in purification, and detection,    -   increased immunogenicity; for example by general        immunostimulation comparable to an adjuvant, or by specific        manipulation of the immunesystem in a certain direction (e.g.        Th1 or Th2), to a certain level of response (higher or lower        than otherwise), or to obtain a certain timing of the immune        response.    -   increased expression level; for instance by enhancing the level        of the expression per se, e.g. by enhancing transcription or        translation levels in a particular expression system, or by        improving the stability of the produced mRNA, or of the protein,        either intra- or extracellularly.    -   providing a linker or spacer region for yet another fusion or        carrier protein, etc.

Examples of such carrier or fusion proteins are well known, forinstance:

-   -   for purification or detection: His-tag, v-Myc-tag,        β-galactosidase (β-gal), maltose binding protein, green        fluorescent protein (GFP), gluthation S-transferase,        streptavidin, biotin, immunoglobulin derived domains (e.g.        Fab-fragments), etc.    -   for improved or adapted immune response: bovine or ovine serum        albumin, keyhole limpet haemocyanin, hsp70, tetanus toxoid, and:        interleukins, cytokines, and hormones that are biologically        active in salmonid fish.

Several of these examples have more than a single effect, for instanceby attaching GFP, both detection and expression is improved.

In an alternative aspect, the invention relates to a polypeptidecomprising a part of an SAV E2 protein, characterised in that said parthas at least the amino acid sequence of SEQ ID NO: 4 and at most theamino acid sequence of SEQ ID NO: 6.

SEQ ID NO: 4 is the consensus sequence derived from the multiplealignment of the fragments aa 158-252 from SAV E2 proteins, alreadydescribed above in Table 1. The alignment and the consensus sequence aredepicted in FIG. 2.

SEQ ID NO: 5 and 6 are consensus sequences derived in a similar way fromSAV E2 proteins, covering aa 139-290, and 111-304, respectively.

Such a polypeptide according to the invention, comprises a part of theSAV E2 protein that is equal or larger than SEQ ID NO: 4, but not largerthan SEQ ID NO: 6. Such polypeptides still comprise the discontinuous VNepitope of SAV E2 protein, through the incorporation of SEQ ID NO: 4.

Polypeptides according to the invention, have a size of between 95 and194 amino acids (respectively the length of SEQ ID NO: 4 and 6), and areselected from the sequence of this region of SAV E2.

Because SEQ ID NO: 4 and 6 are consensus sequences, a certain level ofvariability in the amino acid sequence of the polypeptides according tothis aspect of the invention is within the scope of the invention. Theseconsensus sequences have at least 85% amino acid identity to the regionof aa 158-252 of any of the three SAV subtypes.

One example of a protein according to this aspect of the invention isSEQ ID NO: 5, which runs from aa 139-290 relative to the sequence of SAVE2.

In a preferred embodiment, the polypeptide according to this aspect ofthe invention, comprises SEQ ID NO: 4, with an increased level of aminoacid sequence identity, obtained by replacing one or more of thevariable amino acids (indicated by the symbol X or Xaa, representing anyamino acid) at a certain position in SEQ ID NO: 4, corresponding to acertain position in the SAV E2, by one of the amino acids indicated forthat position in Table 2:

TABLE 2 Preferred embodiments of SEQ ID NO: 4; amino acids to replacethe variable amino acids in SEQ ID NO: 4. position in position in SEQ IDNO: 4 SAV E2 replace X by: 2 159 L or M 18 177 P or L 29 186 I or T 40197 N or S 41 198 D or E 42 199 N, S, or R 47 204 R or K 49 206 S or P59 216 K or R 62 219 S or N 64 221 A or D 65 222 Q, S, or K 66 223 A orE

For both alternatives of the polypeptide of the invention applies thatthe exact borders of the VN epitope of SAV E2 can be further defined bythe skilled person based on the information provided herein, by usingwell known techniques. The current borders indicated according to theinvention, are set at aa 158-252, whereas it was found that fragmentsfrom aa 139-242 (SEQ ID NO: 7, from SAV subtype 3, isolate N3), and fromaa 170-252 (SEQ ID NO: 8, from SAV subtype 3, isolate N3) both do notpossess a functional VN epitope. Consequently the borders of thestrictest region comprising the VN epitope are, on the N-terminal side:between aa 158 and 170, and on the C-terminal side: between 242 and 252.

In an embodiment of the polypeptide of this aspect of the invention, theinvention relates to a protein comprising the polypeptide of theinvention, whereby said protein does not comprise a part of an SAV E2protein comprising said polypeptide and being larger in size than saidpolypeptide.

Similar to the advantageous uses of the protein of the alternativeaspect of the invention, these proteins now provide fusion- orcarrier-proteins comprising the VN epitope of SAV E2 protein that allowfor instance: easier handling, increased immunogenicity, or an increasedexpression level of the VN epitope of the invention.

Techniques to produce such fusion- or carrier proteins, as describedbefore, are well known in the art.

In another aspect, the invention relates to a carrier comprising aprotein according to the invention.

Such carriers according to this aspect of the invention are organic oranorganic (multi-) molecular structures that can advantageously beemployed for instance to improve the stability, the immunogenicity, thedelivery, or the utility as a diagnostic, of the protein according tothe invention. Examples of carrier molecules of use to vaccination andimmunostimulation are: proteins, lipids, carbohydrates; vesicles such asmicelles, liposomes, ISCOM's, dendromers, niosomes, bio-microcapsules,micro-alginates, macrosols; anorganic compounds such asaluminium-hydroxide, -phosphate, -sulphate or -oxide, silica, Kaolin®,and Bentonite®; and host cells and live recombinant carriers.

Carriers, of use for diagnostic purposes comprise for example particlesof silica, latex, or gold; membranes of nylon, PVDF, nitrocellulose, orpaper; and objects such a silicium chip or a micro-titration devices.

Techniques for the incorporation, coupling, and attachment of a proteinaccording to the invention to such carriers are well known in the art.All such embodiments are described in more detail below.

Embodiments of carrier proteins, comprise much the same examples asdescribed above, now providing for instance: easier handling, increasedimmunogenicity, or an increased expression level of the proteinscomprising a polypeptide according to the invention.

Carbohydrates and lipids as carrier for a protein according to theinvention, are for instance lectins, glucans, glycans andlipopolysaccharides, such as lipid A. Techniques for coupling of aprotein according to the invention with such molecules are well known inthe art (e.g. Kubler et al., 2005, J. Org. Chem., vol. 70, p. 6987-6990;Alving, 1991, J. Immunol. Methods, vol. 140, p. 1-13).

Carrier vesicles serve multiple purposes, for instance as stabiliser, asdelivery vehicle and as immunostimulant. For instance ISCOM's are wellknown immunostimulating particles (WO 96/11711), consisting of a mixtureof saponin, a phospholipid and cholesterol, into which an antigen suchas a protein according to the invention can be incorporated.Alternatively, Iscom-matrix particles can be produced. These areIscom-like particles in which the subunit antigen is not integrated butadsorbed.

Carriers for use in oral vaccination, provide both delivery and immunestimulation. Examples are metabolisable substances such asalpha-cellulose or different oils of vegetable or animal origin. Also anattractive way is the use of live feed organisms as carriers; when suchorganisms have taken up or adsorbed the protein according to theinvention, they can be fed to the target fish. Particularly preferredfeed carriers for oral delivery of the protein according to theinvention are live-feed organisms which are able to encapsulate theprotein. Suitable live-feed organisms include plankton-likenon-selective filter feeders, preferably members of Rotifera, Artemia,and the like. Highly preferred is the brine shrimp Artemia sp.

A preferred method of preparing a feed carrier is to feed host cells orcells from an expression system, comprising the protein according to theinvention, to plankton-like non-selective filter feeders preferablymembers of Rotifera, Artemia, and the like. The protein is taken up bythe live feed carrier such as plankton-like non-selective filterfeeders, and these are then administered orally to the fish to beprotected against SAV infection and disease.

A live recombinant carrier (LRC) is a micro-organism such as e.g.bacteria, parasites and viruses, into which additional geneticinformation has been inserted, in this case a nucleic acid, a DNAfragment, or a recombinant DNA molecule, capable of encoding a proteinaccording to the invention (as will be outlined below). Because the LRCreplicates in vivo in the target animal, only relatively little inoculumneeds to be applied, compared to the amount of antigenic protein neededfor a ‘classical’ vaccination. This way, the LRC expresses the antigenof choice directly in the target animal, or in its cells. This providesa favourable presentation to the host's immune system. Alternatively,LRC's can serve as a carrier for the genetic information encoding thedesired antigen, in other words: as a delivery vehicle for a nucleicacid vaccine to the cells of the target animal.

Target organisms inoculated with such LRC's produce an immunogenicresponse against the immunogens of the carrier as well as against theheterologous protein(s) for which the genetic code is additionallycloned into the LRC, e.g. a nucleic acid capable of encoding an SAV E2VN epitope comprised in a polypeptide or a protein according to theinvention. The immune response induced against antigens from the carriermicro-organism boosts the response to the protein according to theinvention.

Also, an LRC with an insert effectively forms a combination vaccine,providing multiple protection in one vaccination.

When the LRC is a virus, such viruses are also called carrier- or vectorviruses. Viruses that can advantageously be used as LRC for theinvention are viruses able to replicate in salmonid fish, for whichsufficient molecular biological information is available to allowcloning and manipulation. Preferred viral LRC's are Alphaviruses, andviruses from the genus Novirhabdo-virus, especially the species viralhemorrhagic septicaemia virus, and infectious haematopoietic necrosisvirus (IHNV). For instance IHNV is a trout pathogen, for which anattenuated viral expression and delivery system for use in salmonids hasbeen described. (WO 03/097090; Biacchesi et al., 2000, J. of Virol.,vol. 74, p. 11247-11253). Deletion of the NV protein from the IHNVgenome attenuates the virus and creates room for insertion of a foreigngene. A preferred LRC is a recombinant IHNV carrying a nucleic acidconstruct capable of encoding a polypeptide or protein according to theinvention. Such an LRC is then administered to target fish for instanceby immersion vaccination.

A host cell comprising a polypeptide, protein, nucleic acid, or LRCaccording to the invention, can also be used as carrier. For instancethe cells of the expression system that was used to produce thepolypeptide or protein according to the invention, or the cells used toproduce an LRC according to the invention may be formulated into apharmaceutical composition, such as a vaccine, and be administered tothe target fish. As with LRC's, antigens from the host cell will providesome additional immunostimulation, having an adjuvating effect.

Anorganic carriers can be coated with, or can adsorb a peptide orprotein according to the invention using well known techniques. Suchloaded carriers are then employed in vaccine formulations forapplication to target fish, or for application in diagnostic assays. Forinstance aluminium hydroxide is a well known vaccine adjuvant.Similarly, particles of silica (glass beads) or gold are commonly usedin diagnostic assays.

Carriers comprising a polypeptide or protein according to the inventioncan also be used in diagnostic assays. For instance membranes or stripsof a suitable material, such as nylon, PVDF, nitrocellulose, or papercan be produced by adsorbing, coating, or blotting the polypeptide orprotein according to the invention. Techniques for e.g. blotting using aliquid flow or electric current are well known in the art. Suchmembranes are then incorporated in a test device or diagnostic kit.Objects may also serve as carrier: for instance a silicium chip for usein BIAcore equipment, or a micro-titration device with wells coated withthe polypeptide or the protein according to the invention, canadvantageously be used.

A diagnostic assay using such a carrier is very favourable for thespecific detection of SAV. In particular because the VN epitope of SAVE2 comprised in a polypeptide, protein, or carrier according to theinvention allows specific detection of al three subtypes of SAV; asdescribed herein, the VN epitope of SAV E2 is much conserved between thethree subtypes, having amino acid identities between 90 and 100%.

Such specific identification of SAV is for instance very useful in thedetermination of the cause of disease; e.g. the aquatic Birnavirusinfectious pancreatic necrosis virus (IPNV) also causes disease andmortality in salmonid fish. As the symptoms of SPDV and IPNV can bedifficult to differentiate, having a sensitive and specific diagnostictest for SAV available greatly improves the possibilities for applyingthe correct measures to the animal husbandry and health care.

It is one of the merits of the invention that the polypeptide, protein,or carrier according to the invention, can also be used to producespecific antibodies against the SAV E2 VN epitope. As a result, it isnow for the first time possible to produce antibodies, monoclonal orpolyclonal, that are almost exclusively directed against the SAV E2 VNepitope. Such antibodies are highly specific for SAV, and highlyeffective in for instance passive immunisations and diagnostic assays(as will be outlined below). Such antibodies are then used e.g. fortherapy, for diagnostics, or for quality assurance purposes.

Therefore in an embodiment the invention relates to a method ofproducing salmonid alphavirus neutralising antibodies, comprising theinoculation of the polypeptide, the protein, or the carrier according tothe invention into an animal, and isolation of antibodies.

Methods of raising and producing antibodies, or antisera comprisingantibodies, as well as the concept of “specific binding” by an antibody,are well-known in the art. For instance antibodies or antiserum againstthe polypeptide, the protein, or the carrier according to the inventioncan be obtained quickly and easily by vaccination of e.g. pigs, poultryor rabbits with the polypeptide, protein, or carrier according to theinvention in e.g. a water-in-oil emulsion followed, after 2-6 weeks, bybleeding, centrifugation of the coagulated blood and decanting of thesera.

Another source of antibodies is the blood or serum of trout or salmonthat have been (naturally) infected with SAV.

Other methods for the preparation of antibodies, which may bepolyclonal, monospecific, or monoclonal (or derivatives thereof) arewell-known in the art. If polyclonal antibodies are desired, techniquesfor producing and processing polyclonal sera are well-known in the art(e.g. Mayer and Walter eds., 1987, Immunochemical Methods in Cell andMolecular Biology, Academic Press, London).

Monoclonal antibodies, reactive against the polypeptide or proteinaccording to the invention can be prepared by immunizing inbred mice bytechniques also known in the art (Kohler & Milstein, 1975, Nature, vol.256, p. 495-497).

In a further aspect, the invention relates to a nucleic acid encodingthe polypeptide or the protein according to the invention.

The term “nucleic acid” is meant to incorporate a molecular chain ofdesoxy- or ribo-nucleic acids. A nucleic acid is not of a specificlength, therefore polynucleotides, genes, open reading frames (ORF's),probes, primers, linkers, spacers and adaptors, consisting of DNA and/orRNA, are included within the definition of nucleic acid. A nucleic acidcan be of biologic and/or synthetic origin. The nucleic acid may be insingle stranded or double stranded form. The single strand may be insense or anti-sense orientation. Modifications in the bases of thenucleic acid may be made, and bases such as Inosine may be incorporated.Other modifications may involve, for example, modifications of thebackbone.

With the term “encoding” is meant: providing the possibility of proteinexpression, i.a. through transcription and/or translation when broughtinto the right context. The right context refers to the promoter, cells,buffer, reaction conditions, etc.

A nucleic acid according to the invention when brought into the rightcontext, is capable of encoding a polypeptide or a protein according tothe invention. Examples of nucleic acids according to the invention havebeen described above.

Methods to isolate a nucleic acid capable of encoding a polypeptide orprotein according to the invention have been described above and arewell-known in the art. For instance the SAV E2 VN epitope contained inpolypeptides as depicted in SEQ ID NO: 1-3 can be used to make orisolate probes or primers. These are then used to screen libraries ofgenomic or mRNA sequences by PCR or hybridization selection. From apositive clone or colony, the VN epitope containing fragment is thenisolated, sub cloned and used e.g. in an expression system.

Alternatively, E2 VN epitopes according to the invention, from other SAVisolates can now conveniently be identified by computerised comparisonsof SEQ ID NO:1-3 in silico to other SAV sequences that may be comprisedin a computer database. For that purpose many computer programs arepublicly available. For instance the suite of BLAST programs (Altschulet al., 1997, Nucleic Acids Res., vol. 25, p. 3389-3402) can be employedto compare SEQ ID NO: 1-3 to expressed sequence tags (EST)- and genomicsequence databases.

Nucleic acids according to the invention also include nucleic acidshaving variations in the nucleotide sequence when compared to SEQ ID NO:1-6. “Variant” nucleic acids may be natural or non-natural variants.Natural variants exist in the various isolates of SAV; non-naturallyoccurring variants may be created by rec DNA techniques.

It is well-known in the art, that many different nucleic acids canencode one and the same protein. This is a result of what is known inmolecular biology as “wobble”, or the “degeneracy of the genetic code”,wherein several different codons or triplets of mRNA will cause the sameamino acid to be attached to the chain of amino acids growing in theribosome during translation. It is most prevalent in the second andespecially the third base of each triplet encoding an amino acid. Thisphenomenon can result in a heterology of about 30% for two differentnucleic acids that still encode the same protein. Thus, two nucleicacids having a nucleotide sequence identity of about 70% can stillencode one and the same protein.

Nucleic acids encoding a polypeptide or protein according to theinvention, can be obtained, manipulated and expressed by standardtechniques in molecular biology, as described above. The tools for suchmanipulations and expressions are carriers for the nucleic acidaccording to the invention.

Therefore a further aspect of the invention relates to a carriercomprising a nucleic acid according to the invention, whereby saidcarrier is selected from the group consisting of a DNA fragment, arecombinant DNA molecule, a live recombinant carrier, and a host cell.These are described in more detail below.

In a preferred embodiment the invention relates to a DNA fragmentcomprising a nucleic acid according to the invention.

A preferred carrier is a DNA plasmid.

The preferred method of obtaining a DNA fragment is by reversetranscription of isolated mRNA by using RT-PCR. PCR techniques arecommonly known, as described above.

An isolated cDNA sequence may be incomplete due to incompletetranscription from the corresponding mRNA, or clones may be obtainedcontaining fragments of the complete cDNA. Various techniques are knownin the art to complete such partial cDNA sequences, such as RACE (rapidamplification of cDNA ends).

In another preferred embodiment the invention relates to a recombinantDNA molecule comprising a nucleic acid, or a DNA fragment according tothe invention, wherein the nucleic acid, or the DNA fragment arefunctionally linked to a promoter.

To construct a recombinant DNA molecule according to the invention, DNAplasmids carrying promoters can advantageously be employed, as describedabove.

In yet another preferred embodiment, the invention relates to a liverecombinant carrier (LRC) comprising a nucleic acid, a DNA fragment, ora recombinant DNA molecule according to the invention. LRC's have beendescribed in detail above.

The DNA fragment, the recombinant DNA molecule, or the LRC according tothe invention may additionally comprise other nucleotide sequences suchas immune-stimulating oligonucleotides having unmethylated CpGdinucleotides, or nucleotide sequences that code for other antigenicprotein, or adjuvating cytokines.

In still another preferred embodiment, the invention relates to a hostcell comprising a nucleic acid, a DNA fragment, a recombinant DNAmolecule, or an LRC, all according to the invention.

A host cell according to the invention may comprise such a nucleic acid,DNA fragment, recombinant DNA molecule, or LRC according to theinvention, stably integrated into its genome, or on an extrachromosomalbody replicating autonomously.

Examples of host cells as carrier, or for the purpose of expression of apolypeptide or protein according to the invention have been describedabove. In the use as a vaccine, the host cell may be live orinactivated, depending on the desired effect. Many physical and chemicalmethods of inactivation of cells are known in the art; examples ofphysical inactivation are by heating, or by radiation, e.g. with UV,X-rays or gamma-radiation. Examples of inactivating chemicals areβ-propiolactone, glutaraldehyde, β-ethylene-imine and formaldehyde. Whena method of inactivation is to be applied, the skilled person knows thisrequires optimisation, in order not to disturb the immunogenicity of thepolypeptide, protein, carrier, or nucleic acid that is to be delivered,comprised in the host cell, to the target animal.

Preferred use of a nucleic acid, a DNA fragment, a recombinant DNAmolecule, an LRC, or a host cell according to the invention is inexpression and delivery of the SAV E2 VN epitope. One way to achievethat is through DNA vaccination. DNA plasmids carrying a nucleic acid, aDNA fragment, a recombinant DNA molecule according to the invention canbe administered to a salmonid fish as described above. Such methods arewell-known in the art.

Nucleic acid vaccines (or gene- or genetic-vaccines as they are called)may require a targeting- or a delivery vehicle other than an LRC totarget or protect it, or to assist in its uptake by (the cells of) thehost. Such vehicles may be biologic or synthetic, and are for instancebacteriophages, virus-like particles, liposomes, or micro-, powder-, ornano particles.

DNA vaccines can easily be administered through intradermal applicatione.g. using a needle-less injector such as a GENEGUN®. This way ofadministration delivers the DNA directly into the cells of the animal tobe vaccinated. A preferred amount of a nucleic acid, a DNA fragment, ora recombinant DNA molecule according to the invention, comprised in apharmaceutical composition according to the invention (as outlinedbelow) is in the range between 10 pg and 1000 μg. Preferably, amounts inthe range between 0.1 and 100 μg are used. Alternatively, fish can beimmersed in solutions comprising e.g. between 10 pg and 1000 μg/ml ofthe DNA to be administered. All these are well-known in the art.

Similarly, a targeting- or delivery vehicle comprising a nucleic acid, aDNA fragment, or a recombinant DNA molecule according to the invention,is within the scope of a carrier according to the invention.

These uses will result in nucleic acid being delivered, or protein beingexpressed inside the target organism or its cells.

The medical uses of a polypeptide, protein, carrier, or nucleic acidaccording to the invention have been described above. In essence this isthe vaccination of fish against SAV, in order to prevent or ameliorate,infection or disease, by interfering with the establishment and/or withthe progression of an SAV infection, or with the progression of clinicalsymptoms of SAV induced disease.

These medical uses are put to practice in aquatic animal health caree.g. by administering to a salmonid fish a polypeptide, protein,carrier, or nucleic acid according to the invention.

Therefore, another aspect of the invention relates to a pharmaceuticalcomposition comprising the polypeptide, protein, carrier, or nucleicacid according to the invention, and a pharmaceutically acceptablecarrier.

In an embodiment, the pharmaceutical composition of the inventionrelates to a vaccine comprising a polypeptide, a protein, a carrier, ora nucleic acid according to the invention, and a pharmaceuticallyacceptable carrier.

In a further aspect, the invention relates to the polypeptide, protein,carrier, or nucleic acid according to the invention, for use as amedicament for fish.

In a further aspect, the invention relates to the use of a polypeptide,protein, carrier, or nucleic acid according to the invention, for themanufacture of a medicament for fish.

In a further aspect, the invention relates to a method of vaccination offish by administering to such organism a polypeptide, protein, carrier,or nucleic acid according to the invention, in a pharmaceuticallyeffective amount and in a pharmaceutically acceptable carrier.

A “pharmaceutically effective amount” is described in detail below.

In a further aspect the invention relates to a method of producing avaccine for fish, by admixing the polypeptide, protein, carrier, ornucleic acid according to the invention with a pharmaceuticallyacceptable carrier.

A “pharmaceutically acceptable carrier” can e.g. be water, saline, or abuffer suitable for the purpose. In a more complex form the formulationmay comprise an emulsion which itself comprises other compounds, such asa cytokine, an adjuvant, an additional antigen, etc.

The vaccine according to the invention can be used both for prophylacticand for therapeutic treatment.

In a preferred embodiment the vaccine according to the inventionadditionally comprises an adjuvant.

An adjuvant is an immunostimulatory substance boosting the immuneresponse of the host in a non-specific manner. Many different adjuvantsare known in the art. Examples of adjuvants frequently used in fishfarming are muramyldipeptides, lipopolysaccharides, several glucans andglycans and CARBOPOL® (a homopolymer). An extensive overview ofadjuvants suitable for fish vaccines is given in the review paper by JanRaa (1996, Reviews in Fisheries Science, vol. 4, p. 229-288).

Suitable adjuvants are e.g. water in oil (w/o) emulsions, o/w emulsionsand w/o/w double-emulsions. Oil adjuvants suitable for use in w/oemulsions are e.g. mineral oils or metabolisable oils. Mineral oils aree.g. BAYOL® MARCOL® and DRACOL®; metabolisable oils are e.g. vegetableoils, such as peanut oil and soybean oil, or animal oils such as thefish oils squalane and squalene. Alternatively a vitamin E (tocopherol)solubilisate as described in EP 382,271 may advantageously be used.

Very suitable o/w emulsions are e.g. obtained starting from 5-50& w/wwater phase and 95-50% w/w oil adjuvant, more preferably 20-50% w/wwater phase and 80-50% w/w oil adjuvant are used.

The amount of adjuvant added depends on the nature of the adjuvantitself, and information with respect to such amounts provided by themanufacturer.

In a preferred embodiment the vaccine according to the inventionadditionally comprises a stabiliser.

A stabilizer can be added to a vaccine according to the invention e.g.to protect it from degradation, to enhance the shelf-life, or to improvefreeze-drying efficiency. Useful stabilizers are i.a. SPGR (Bovarnik etal., 1950, J. Bacteriology, vol. 59, p. 509), skimmed milk, gelatine,bovine serum albumin, carbohydrates e.g. sorbitol, mannitol, trehalose,starch, sucrose, dextran or glucose, proteins such as albumin or caseinor degradation products thereof, and buffers, such as alkali metalphosphates.

In addition, the vaccine may comprise one or more suitablesurface-active compounds or emulsifiers, e.g., SPAN® or TWEEN®.

The vaccine may also comprise a so-called “vehicle”. A vehicle is acompound to which the polypeptide or the protein according to theinvention adheres, without being covalently bound to it. Such vehiclesare i.a. bio-microcapsules, micro-alginates, liposomes and macrosols,all known in the art. A special form of such a vehicle is an Iscom,described above.

It goes without saying that admixing other stabilizers, carriers,diluents, emulsions, and the like to vaccines according to the inventionare also within the scope of the invention. Such additives are forinstance described in well-known handbooks such as: “Remington: thescience and practice of pharmacy” (2000, Lippincot, USA, ISBN:683306472), and: “Veterinary vaccinology” (P. Pastoret et al. ed., 1997,Elsevier, Amsterdam, ISBN: 0444819681).

For reasons of e.g. stability or economy a composition according to theinvention may be freeze-dried. In general this will enable prolongedstorage at temperatures above zero ° C., e.g. at 4° C. Procedures forfreeze-drying are known to persons skilled in the art, and equipment forfreeze-drying at different scales is available commercially.

Therefore, in a preferred embodiment, the vaccine according to theinvention is in a freeze-dried form.

To reconstitute a freeze-dried composition, it is suspended in aphysiologically acceptable diluent. Such a diluent can e.g. be as simpleas sterile water, or a physiological salt solution. In a more complexform the freeze-dried vaccine may be suspended in an emulsion e.g. asdescribed in EP 1,140,152.

A vaccine according to the invention may take any form that is suitablefor administration in the context of aqua-culture farming, and thatmatches the desired route of application and desired effect. Preparationof a vaccine according to the invention is carried out by meansconventional for the skilled person.

Preferably the vaccine according to the invention is formulated in aform suitable for injection or for immersion vaccination, such as asuspension, solution, dispersion, emulsion, and the like. Commonly suchvaccines are prepared sterile.

Target animal for the vaccine according to the invention is a fish,preferably a salmonid fish, more preferably a rainbow trout(Oncorhynchus mykiss) or an Atlantic salmon (Salmo salar L.)

The dosing scheme of the application of a vaccine according to theinvention to the target organism can be in single or multiple doses,which may be given at the same time or sequentially, in a mannercompatible with the dosage and formulation, and in such an amount aswill be immunologically effective.

It is well within the capacity of the skilled person to determinewhether a treatment is “immunologically effective”, for instance byadministering an experimental challenge infection to vaccinated animals,and next determining a target animals' clinical signs of disease,serological parameters, or by measuring reisolation of the pathogen.

What constitutes a “pharmaceutically effective amount” for a vaccineaccording to the invention that is based upon a polypeptide, a protein,a carrier, or a nucleic acid according to the invention, is dependent onthe desired effect and on the target organism. Determination of theeffective amount is well within the skills of the routine practitioner.

A preferred amount of a polypeptide or a protein according to theinvention, comprised in a pharmaceutical composition according to theinvention, is between 1 ng and 1 mg per animal dose. More preferably theamount is between 10 ng and 100 μg/dose, even more preferably between100 ng and 10 μg/dose. A dose exceeding 1 mg, although immunologicallyvery suitable, will be less attractive for commercial reasons.

A preferred amount of a nucleic acid, a DNA fragment, or a recombinantDNA molecule according to the invention, comprised in a pharmaceuticalcomposition according to the invention, has been described above.

A preferred amount of a live recombinant carrier according to theinvention, comprised in a vaccine according to the invention, isdependent on the characteristics of the carrier micro organism used.Such an amount is expressed for instance as plaque forming units (pfu),colony forming units (cfu) or tissue culture infective dose 50%(TCID₅₀), depending on what is a convenient way of quantifying the LRCorganism. For instance for a live viral vector a dose range between 1and 10¹⁰ plaque forming units (pfu) per animal dose may advantageouslybe used; preferably a range between 10² and 10⁶ pfu/dose.

A preferred amount of a host cell according to the invention, comprisedin a vaccine according to the invention, is between 1 and 10⁹ host cellsper animal dose. More preferably between 10 and 10⁷ cells/dose are used.

Many ways of administration can be applied, all known in the art. Thevaccines according to the invention are preferably administered to thefish via injection, immersion, dipping or per oral. The protocol for theadministration can be optimized in accordance with standard vaccinationpractice. An overview of fish vaccination by Bowden et al. (FisheriesResearch Service Marine Laboratory, Aberdeen, Scotland) is available asan Industry Report of 27-3-2003.

Preferably the vaccine is administered via immersion or per oral. Thisis especially efficient in case of the use of such vaccines in thesetting of commercial aqua-culture farming.

Preferred embodiments on the use of carriers in oral vaccination havebeen described above.

The age, and therefore the weight, of the fish to be vaccinated is notcritical, although it is evidently favourable to vaccinate against SAVas early as possible to prevent a field infection. Juvenile salmonidscan be vaccinated already at 0.2 grams, and but before reaching 5 gramsof weight. Fish having a weight of less than 0.5 grams however areassumed to be insufficiently immune competent. Therefore, in practice,one would try to vaccinate fish having a weight of between 0.5 and 5grams. Since it is one of the merits of the present invention that it isnow possible to perform early diagnosis of SAV, control measurementssuch as sanitation can be developed in order to postpone or reduceoutbreaks in the geographical area, until fish have been vaccinated.

It is highly efficient to formulate a vaccine according to the inventionas a combination-vaccine, that is by combining a polypeptide, protein,carrier, or nucleic acid according to the invention, with at least oneother fish-pathogenic micro organism or virus, with an antigen of suchmicro organism or virus, or with a nucleic acid encoding such anantigen.

Therefore, in a preferred embodiment, the vaccine according to theinvention is a combination vaccine.

The advantage of such a combination vaccine is that it not only providesprotection against SAV, but also against other diseases, while only onevaccination manipulation is required, thereby preventing needless stressto the animals as well as time- and labour costs.

In a more preferred embodiment the combination vaccine according to theinvention comprises at least one other micro organism or virus that ispathogenic to fish, preferably to salmonids, or one other antigen from avirus or micro organism pathogenic to fish, or a nucleic acid encodingsaid other antigen.

Therefore, in an even more preferred embodiment of the combinationvaccine according to the invention, the other micro organism or virus,or the antigen, or the nucleic acid encoding said other antigen, isselected from the group consisting of Aeromonas salmonicida subsp.salmonicida, Vibrio anguillarum, V. anguillarum serovar O1 and serovarO2, V. salmonicida, Moritella viscosa (=V. viscosus), Photobacteriumdamselae subspecies piscicidae, Tenacibaculum maritimum, Yersiniaruckeri, Piscirickettsia salmonis, Renibacterium salmoninarum,Lactococcus garvieae, Flavobacterium sp., Flexibacter sp., Streptococcussp., Lactococcus garviae, Edwardsiella tarda, E. ictaluri, InfectiousPancreatic Necrosis Virus, Infectious Salmon Anaemia virus, NervousNecrosis Virus, and Heart and skeletal muscle inflammation.

The disease Heart and skeletal muscle inflammation (HSMI) is a recentlydescribed salmonid disease of viral origin (Kongtorp et al., 2004, J. ofFish diseases, vol. 27, p. 351-358).

The vaccines according to the invention, described above, contribute toactive vaccination, i.e. they trigger the host's defense system.Alternatively, virus neutralising antibodies can be raised against thepolypeptide, the protein, or the carrier according to the invention, asoutlined above. Such VN antibodies can then be administered to the fish.This method of vaccination, so-called passive vaccination, is thevaccination of choice when an animal is already infected, and there isno time to allow the natural immune response to be triggered. It is alsothe preferred method for vaccinating animals that are prone to suddenhigh infection pressure. The administered VN antibodies neutralise SAV,which has the great advantage that it decreases or stops theestablishment or progression of an SAV infection almost immediately,independent of the fish's immune status.

Therefore, in an alternate aspect, the invention provides a vaccinecomprising salmonid alphavirus neutralising antibodies.

A vaccine can also be prepared using antibodies prepared from eggs ofchickens that have been vaccinated with a vaccine according to theinvention (IgY antibodies).

Preferably a vaccine for oral administration of the antibodies isprepared, in which the antibodies are mixed with an edible carrier suchas fish feed.

In another aspect, the invention relates to a diagnostic test kitcomprising a polypeptide, a protein, a carrier, or a nucleic acidaccording to the invention.

As mentioned above, mortality after SAV infection can be up to 60%.Thus, for efficient protection and control measures against SAV and itsinduced disease, a quick and specific diagnosis of SAV is important.

Therefore it is another objective of this invention to providediagnostic tools suitable for the detection of SAV.

A diagnostic test kit for the detection of antigenic material comprisingan SAV E2 VN epitope according to the invention is suitable and specificfor the detection of all SAV subtypes.

Such a test may e.g. comprise a standard antigen ELISA test, wherein thewells of an ELISA plate are coated with an antibody directed against theSAV E2 VN epitope. Such antibodies are reactive with the SAV E2 VNepitope as comprised in the polypeptide, the protein, or the carrieraccording to the invention, and can be obtained as described above.After incubation with the material to be tested, labelled antibodiesreactive with the SAV E2 VN epitope are added to the wells. A colourreaction then reveals the presence of bound antigenic material of SAV.Protocols for labelling antibodies by coupling of a fluorescent group tothe immunoglobulin or another marker, are well known in the art.

Also, a diagnostic test kit for the detection in a sample or in ananimal serum of antibodies reactive with the SAV E2 VN epitope accordingto the invention is suitable and specific for the detection of an immuneresponse against SAV of any of the subtypes.

Such a test may e.g. comprise a standard antigen ELISA test, wherein thewells of an ELISA plate are coated with an antibody directed against theSAV E2 VN epitope. Such antibodies are reactive with the SAV E2 VNepitope as comprised in the polypeptide, the protein, or the carrieraccording to the invention, and can be obtained as described above.After incubation with the material to be tested, labelled antibodiesreactive with the SAV E2 VN epitope are added to the wells. A colourreaction then reveals the presence of bound antigenic material of SAV.Protocols for labelling antibodies by coupling of a fluorescent group tothe immunoglobulin or another marker, are well known in the art.

Therefore, in an embodiment the invention relates to diagnostic testkits for the detection of antibodies reactive with the SAV E2 VNepitope. Such test kits comprise the polypeptide, the protein, or thecarrier according to the invention.

The design of such an immunoassay may vary. For example, the immunoassaymay also be based upon competition, in stead of on direct binding.Furthermore, such tests may also use particulate or cellular material,in stead of the solid support of a device. The detection of theantibody-antigen complex formed in the test may involve the use oflabelled antibodies, wherein the labels may be, for example, enzymes orfluorescent-, chemo luminescent-, radio-active- or dye molecules.

Suitable methods for the detection of antibodies reactive with the SAVE2 VN epitope according to the invention in a sample include theenzyme-linked immunosorbent assay (ELISA), immunofluorescence test (IFT)and Western blot analysis.

A quick and easy diagnostic test for diagnosing the presence or absenceof SAV is a PCR test as described above, comprising primers specificallyhybridising to a nucleic acid in a test sample that is similar to anucleic acid according to the invention. Such primers are for instancethe following two, giving a product of 188 nucleotides, from within theSAV E2 VN epitope of the invention:

FWD: 5′-TTGACGTGTACGACGCTCTG-3′ (SEQ ID NO: 9)REV: 5′-AACCGGCTCCTCACACGTAAAC-3′ (SEQ ID NO: 10)

The nucleic acid, DNA fragment, recDNA molecule, or carrier according tothe invention can advantageously be used in such PCR test as a positivestandard.

Alternatively, such a diagnostic test may use the nucleic acid, DNAfragment, recDNA molecule, or carrier according to the invention, in aset up using hybridisation without amplification to e.g. a membrane or adevice.

The invention will now be further described with reference to thefollowing, non-limiting, examples.

EXAMPLES Example 1 Cloning

An SPDV E3E2 sequence was obtained from an SAV subtype 3 isolate: anSPDV infected Atlantic salmon was sampled from an aquaculture farm inNorway (Mølsvik). From its heart tissue a total RNA extract was preparedusing an ABSOLUTELY RNA MINIPREP KIT(Stratagene), according to themanufacturer's instructions. From the cDNA prepared, the total E3E2coding region was amplified with RT-PCR primers. The primers used were:

For the reverse transcription, the following primer was used:

RTE2: (SEQ ID NO: 11) 5′-CCGCGCGAGCCCCTGGTATGCAACACAGTGC-3′Next, high fidelity PCR amplification was performed, using pfu turbopolymerase (Stratagene), with the primer set:

RT-E2 Xhol: (SEQ ID NO: 12) 5′-ATACCAGGGGCTCGCGCCTCGAGACCCTACTTG-3′PCR-E3 Hindlll: (SEQ ID NO: 13) 5′-GATGCCATAAGCTTGACACGCGCTCCGGCCCTC-3′Finally the sequence of the E3E2 gene was determined by sequencing withthese two PCR primers, and two internal primers:

PD5: (SEQ ID NO: 14) 5′-CGTCACTTTCACCAGCGACTCCCAGACG-3′ PD3:(SEQ ID NO: 15) 5′-GGATCCATTCGGATGTGGCGTTGCTATGG-3′Sequencing was performed with BIG DYE 3.1® (Applied Biosystems),according to the manufacturer's instructions.

The complete nucleotide sequence for the E2 encoding region of theMølsvik SAV isolate will be published under accession number DQ 195447,but for the invention all relevant sequences have been indicated herein.

Once verified by sequencing, the E3E2 PCR product was digested overnightwith the restriction enzymes XhoI and HindIII, and cloned in thecorresponding unique sites of the pET30a(+) vector (Novagen) leading tothe construct pET30a/E3E2.

Subcloning of Regions Derived from SAV E2:

From the Mølsvik SAV E2 sequence, various shorter sequences wereobtained using a subcloning strategy, in which PCR of a set of primershybridising at various positions on the E2 gene was employed. The PCRprimers used to amplify fragments of the SAV E2 encoding region (usingthe construct pET30a/E3E2 as template) are listed in Table 3, and weredesigned as follows:

-   -   forward primers start with 3-5 random A or T nucleotides,        followed by an NdeI restriction site. Next come 16 to 20        matching nucleotides, the length is optimised depending on the        specific local sequence, and selected to start the PCR fragment        at a desired nucleotide. Primers are 24 to 30 nucleotides in        total.    -   reverse primers also comprise 3-5 random at nucleotides, but are        followed by an NcoI restriction site, and then 16-20 specific        nucleotides.

TABLE 3 Primers used herein, for making fragments of SAV E2. expressedE2 fragment Forward primer starts at aa SEQ ID NO: Frag5-fwd 111 16 F1139 17 F2 158 18 F3 177 19 expressed E2 fragment Reverse primer ends ataa SEQ ID NO: Frag5-rev 304 20 R1 290 21 R2 270 22 R3* 252 23 R4 231 24

Fragments produced in PCR were digested overnight with NdeI and NcoI,and cloned into the pET30a(+) bacterial expression plasmids (Novagen).

Because the NdeI restriction site incorporates an ATG start codon,expression started on this Methionine.

Cloned sequences were verified by DNA sequencing using the standardpET-T7 promoter primer: 5′-AATACGACTCACTATAGGG-3′ (SEC) ID NO: 25) andstandard T7 terminator primer: 5′-GCTAGTTATTGCTCAGCGG-3′ (SEQ ID NO: 26)in order to obtain overlapping contigs covering the whole clonedsequence.

Subscloned fragments of SAV E2 were expressed in E. coli, and theprotein fragments produced were used in in vivo and in vitro experimentsto test the SAV E2 VN epitope of the invention.

Recombinant proteins were also expressed as fusion proteins using thepET30a(+) plasmids, by cloning these in frame with a 6×His tag or aβ-gal gene (LacZ), inserted into a pET30a(+) plamid.

In order to construct the pET30a/LacZ vector, the lacZ gene contained inthe vector pVAX1/LacZ (Invitrogen) was amplified by PCR using theprimers:

LacZ′_Ncol:  SEQ ID NO: 27 5′-GTATGCCCATGGAACGTCGTTTTACAACGTCGTG-3′LacZ′_Xhol:  SEQ ID NO: 28 5′-GTCTCGCTCGAGTTATTTTTGACACCAGACCAACTGG-3′

Following overnight digestion with NcoI and XhoI, the digested PCRproduct was cloned into the corresponding restriction sites of thepET30a(+) plasmid. Digestion of this pET30a/LacZ construct withNdeI/NcoI allowed the in frame insertion of the described E2subfragments digested with NdeI/NcoI.

Example 2 Expression

Expression of the various E2 fragments was done in BL21 Rosetta 2 E.coli cells. The pET3a plasmids containing the various E2 gene fragmentswere used to transform Rosetta 2 cells (Novagen), according to thesuppliers instructions. An overnight pre-culture (20 ml) of transformedE. coli, was used next day to inoculate 800 ml cultures with 5 mlpre-culture. When the OD600 was at approximately 0.7, then IPTG wasadded to 1 mM final concentration for induction of expression. This wascultured for another 2 hours. Then cultures were centrifuged at 7000 rpmfor 5 min, and bacterial cell pellet was kept at −80° C. until use.

Purification:

To purify the inclusion bodies, two buffers were used:

buffer A=50 mM tris/HCl pH8.00+2 mM EDTA

buffer B=buffer A+1% (final concentration) Triton X-100 TRITON X-100®

Then, for the cell pellet of an 800 ml culture: the cell pellet wasresuspended in 50 ml of buffer A and add freshly prepared lysozyme (froma stock at 10 mg/ml) to a final concentration of 100 μg/ml. 25 ml ofbuffer B was then added and 10 μl of BENZONASE® (MERCK). This wasincubated at 30° C. for 15 min. with gentle shaking for removal of DNA.

Next 100 ml buffer B was added and the mixture was sonicated for 4×30seconds. The sonicate was centrifuged at 12.000×g for 20 min. at 4° C.The inclusion body pellet was washed with 250 ml of buffer B, and with200 ml of PBS. Finally the pellet containing the rec E2 proteinfragments were resuspended in 40 ml (4×10 ml) of PBS, and kept at −80°C. until use.

Recombinant E. coli proteins were quantified by scanning and analysis ofsamples run on SDS-PAA gels that had been stained with Coomassie bb., inrelation to lanes with marker protein, according to standard procedures.

Example 3 Testing and Use

Monoclonal Antibody:

For testing the recombinant SAV E2 protein fragments, an SAVneutralising monoclonal antibody was used. This had been produced byimmunising Balb/c mice with 10¹⁰ SAV virus strain S49P in Freund'scomplete adjuvant, obtained from an polyethylene glycol concentratedCHSE-214 cell culture supernatant. Booster injections were given, withFreund's incomplete adjuvant, and without adjuvant, using standardtechniques. Mice spleen cells were fused with Sp₂O tumour cells, andresulting hybridomas were selected in HAT medium. Monoclonal antibodies(Mab's) produced were selected for binding to SAV virus on SAV infectedCHSE-214 cells by immunofluorescence using standard techniques.Positives were tested in a virus neutralisation assay. The hybridomacells producing VN positive Mab's were scaled up by making ascites inmice using standard procedures.

Immunofluorescence assay (IFA):

IFA was performed on SAV infected CHSE-214 cells, and on pET plasmidtransfected E. coli cells expressing the rec protein of interest, withproper positive and negative controls. Cells were fixed in ethanol96%:acetone 1:1 at −20° C. for 30 minutes, and rinsed 3 times with PBST(PBS 1×+0.05% TWEEN 20. The primary antibody, diluted in PBST was addedand incubated 1 hour at room temperature. Next plates were rinsed withPBST, and the second antibody was applied: an FITC conjugated anti-mouseantibody. After incubation and wash, each well was scored throughobservation with an immunofluorescence microscope with the appropriateUV light filter. The infected cells reacting positively with the primaryantibody appeared fluorescent.Immuno-blotting:

For Western blotting samples were run on 10% SDS/PAA gels (NUPAGE®,NOVEX), and blotted onto nitrocellulose according to the manufacturersinstructions. For dot blot, 5 μl samples were spotted on membranesdirectly with a pipette, and left to dry for 5-10 minutes. Next, themembrane was blocked with Tris buffered saline+0.05% Tween 20 (TBST)+5%skimmed milk, 0/N at 4° C. The membrane is incubated with the primaryantibody, diluted to the appropriate concentration in TBST+1% skimmedmilk, for 1 hr at room temperature. Then after 3 washes in TBST, themembrane is incubated with the secondary antibody, an alkalinephosphatase conjugated Goat anti-mouse IgG in TBST. After washing withTBST, alkaline phosphatase colouring reaction was performed withAlkaline Phosphatase Conjugate Substrate Kit according to themanufacturer's instructions (BioRad). Reaction was stopped withdistilled water, membranes were dried and digitized.

Immunofluorescence Assay (IFA):

IFA was performed on SAV infected CHSE-214 cells, and on pET plasmidtransfected E. coli cells expressing the rec protein of interest, withproper positive and negative controls. Cells were fixed in ethanol96%:acetone 1:1 at −20° C. for 30 minutes, and rinsed 3 times with PBST(PBS 1×+0.05% Tween 20. The primary antibody, diluted in PBST was addedand incubated 1 hour at room temperature. Next plates were rinsed withPBST, and the second antibody was applied: an FITC conjugated anti-mouseantibody. After incubation and wash, each well was scored throughobservation with an immunofluorescence microscope with the appropriateUV light filter. The infected cells reacting positively with the primaryantibody appeared fluorescent.

Immuno-Blotting:

For Western blotting samples were run on 10% SDS/PAA gels (NuPAGE®,Novex), and blotted onto nitrocellulose according to the manufacturersinstructions. For dot blot, 5 μl samples were spotted on membranesdirectly with a pipette, and left to dry for 5-10 minutes. Next, themembrane was blocked with Tris buffered saline+0.05% Tween 20 (TBST)+5%skimmed milk, 0/N at 4° C. The membrane is incubated with the primaryantibody, diluted to the appropriate concentration in TBST+1% skimmedmilk, for 1 hr at room temperature. Then after 3 washes in TBST, themembrane is incubated with the secondary antibody, an alkalinephosphatase conjugated Goat anti-mouse IgG in TBST. After washing withTBST, alkaline phosphatase colouring reaction was performed withAlkaline Phosphatase Conjugate Substrate Kit according to themanufacturer's instructions (BioRad). Reaction was stopped withdistilled water, membranes were dried and digitized.

Results of immunoblotting with the polypeptides and proteins accordingto the invention is presented in FIGS. 2 and 3:

FIG. 5 shows a dot blot of various polypeptides and proteins accordingto the invention, stained with an SAV neutralising monoclonal.

Positive recognition was found for:

-   -   the positive control, full length SAV E2 protein,    -   polypeptides F1R1, F1R2, F1R3, and F2R3*

Negative response was detected for:

-   -   the negative control SAV E1 protein    -   the polypeptides F1R4 and F3R3

This ratio of positive and negatives was observed both when the sampleshad been denatured in a sample buffer containing β-mercaptoethanol anddithiothreitol, and were boiled before spotting (upper panel), as whenthe samples were taken up in a non-denaturing sample buffer, and werenot boiled (lower panel).

FIG. 6 shows a Western blot of a polypeptide according to the inventionmade with primers F1 and R3, thus covering the part of SAV E2 from aa139-290; and of a protein according to the invention, the SAV E2fragment made with primers F2 and R3, thus spanning aa 158-252, in afusion construct with β-gal protein. The blot was incubated with an SAVneutralising Mab, and several specific bands were detected.

Bands detected in the F1R3 lanes are at 22 and 44 kDa, probably amonomer and a dimer form of the polypeptide.

NB: although the calculated weight of the polypeptide is only 15 kDa, onthe gel this band runs slower, most likely because of the presence ofdisulfide bridges that were not completely denatured in the samplepreparation.

The F2R3-βgal lanes show a band of approximately 126 kDa, representingthe fusion protein of F2R3 (10 kDa) and β-gal (116 kDa).

VN Assay:

Equal volumes of a dilution of the SAV neutralising Mab and of 100TCID₅₀ of an SAV sample in EMEM culture medium with 2% serum, were mixedin a micro titration plate and incubated 2 hours at 20° C. SAV isolatesfrom all subtypes were used in VN assay (F93-125 [subtype 1], S49P [2],and PD03-08 [3]). Next CHSE-214 cells in EMEM culture medium with 5%serum were added to a density of 2.5×10⁵/ml. The plates were incubatedin CO₂ incubators for 5-7 days at 15° C.

After 5-7 days the plates were read by light microscopy, identifyingcells with cytopathic effect (cpe), indicating the dilution of the Mabnot able to completely neutralise the test virus. Cpe was positive whenclumps of cells were observed that had rounded off, and became morerefringent.

Alternatively cells were fixed in ethanol/acetone, rinsed with PBST, andstained in IFA as described, while using as primary antiserum, apolyclonal rabbit antiserum raised against SPDV E1 protein (serum AB06).

In a typical experiment, the results of a VN test on F93-195, andPD03-08, read by cpe showed full viral neutralisation by the SAVneutralising Mab ascites up to a dilution of 32.000. This was confirmedby IFA.

Animal trial:

In an animal trial juvenile Atlantic salmon were vaccinated withproteins according to the invention. Proteins used were F2R3 fused toβ-gal, and F1R3 fused to a 6× Histidine tag. In short:

proteins were expressed in E. coli and purified as described. Proteinwas formulated to 30% water-in-oil emulsion with Montanide ISA 763A.Animals were vaccinated with 150 μg/dose and boosted with 100 μg/dose.Positive control was the commercial inactivated SPDV vaccine NORVAX®COMPACT PD. Negative controls were: saline, emulsion without protein,and β-gal without fusion.

Test animals were Atlantic salmon smolts of circa 40 grams, that wereacclimatised in the test facility for 1 week, and were then vaccinatedintra-peritoneal using standard procedure. Regular blood sampling wasapplied. After 5 weeks the fish received the booster vaccination andafter an additional 3 weeks the fish will be challenged using a dose of10³ TCID₅₀/animal of PD03-08 (SAV subtype 3). Protective capacity willbe scored by analysing clinical signs of disease, serology, grosspathology and histology.

Antibody Elisa:

An Elisa test was set up, to detect anti-SAV antibodies in salmon serum.General procedure was as described above; briefly: an SAV neutralisingMab was coated to micro titration wells, incubated with a standard SPDVsample and washed. Salmon serum was applied in PBST+1% skimmed milk, ina serial dilution in duplo. Appropriate positive and negative controlswere applied. Incubation was overnight at 4° C. next the plates werewashed and incubated with a rabbit polyclonal anti-salmon serum. Finallya third antibody is incubated: goat anti-rabbit, conjugated to HRP.Finally colorimetric detection was done by using an HRPsubstrate andH₂O₂, according to the manufacturer's instructions.

Typically, SAV antibodies could be detected with good specificity andsensitivity in sera from SPDV infected salmon.

BRIEF DESCRIPTION OF THE DRAWINGS:

FIG. 1:

Graphical representation of the length and position of the variousprotein sequences described herein, relative to the correspondingregions of the full length SAV E2 protein.

FIGS. 2, 3:

Multiple alignment of the regions of aa 158-252, 139-290, or 111-304respectively, of several SAV E2 proteins. Entries are identified byaccession number, and are described in Table 1. Reference strain is SPDVisolate N3 (acc. nr: AAU01400.1). The consensus sequence from themultiple alignment of these regions equals SEQ ID NO: 4, 5, or 6.

Numbers above the sequences indicate the number of the amino acidscorresponding to SAV E2; numbers below the consensus sequences indicatethe total length of the polypeptides.

The invention claimed is:
 1. An isolated salmonoid alphavirus (SAV)polypeptide comprising an amino acid sequence having at least 90% aminoacid identity to SEQ ID NO: 1 or SEQ ID NO: 2, wherein the polypeptideis at least 95 and at most 166 amino acids in size.
 2. A recombinantprotein comprising a polypeptide according to claim 1, wherein, exceptfor the polypeptide of claim 1, said protein is not a salmonidalphavirus (SAV) E2 protein and said recombinant protein being more than166 amino acids in size.
 3. A carrier comprising a protein according toclaim
 2. 4. A method of producing salmonid alphavirus neutralisingantibodies, comprising the steps of a) administering a polypeptideaccording to claim 1 to an animal, b) obtaining serum from the animal,and c) isolating antibodies reactive with the polypeptide from theserum.
 5. An isolated nucleic acid encoding a polypeptide according toclaim
 1. 6. Carrier comprising a nucleic acid according to claim 5,whereby said carrier is selected from the group consisting of a DNAfragment, a recombinant DNA molecule, a live recombinant carrier, and ahost cell.
 7. A vaccine comprising a polypeptide according to claim 1and a pharmaceutically acceptable carrier.
 8. A diagnostic kitcomprising a polypeptide according to claim 1.